The present invention is directed to the fields of cancer and molecular genetics. Specifically, the present invention is directed to the determination of susceptibility to breast cancer and the diagnosis of invasive breast cancer. More specifically, the present invention is directed to a mutation in estrogen receptor alpha (ER) and its association with breast cancer.
Invasive breast cancer (IBC) is one of the most common and lethal malignant neoplasms affecting women, especially in Western cultures. The majority of IBCs are thought to develop over long periods of time from certain preexisting benign lesions. There are many types of benign lesions in the human breast, and only a few appear to have significant premalignant potential. The most important premalignant lesions recognized today are referred to as atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH), ductal carcinoma in situ (DCIS), and lobular carcinoma in situ (LCIS). Although DCIS and LCIS possess some malignant properties, such as loss of growth control, they lack the ability to invade and metastasize and, in this sense, are premalignant.
A skilled artisan is aware that investigation of the role of the estrogen receptor in carcinomas is described by Watts et al., J. Steroid Biochem. Molec. Biol. 41 (3), 529 (1992); Scott et al., J. Clinic. Invest. 88, 700 (1991); Ince et al., J. Bio. Chem. 268, 14026 (1993); Fuqua et al., Can. Res. 52, 43 (1992); McGuire et al., Mol. Endocr. 5, 1571 (1991); Castles et al., Can. Res. 53, 5934 (1993); and Weigel and deConinck, Can. Res. 53, 3472 (1993). Furthermore, description of the estrogen receptor mRNA may be found in Keaveney et al., J. Mol. Endocr. 6, 111 (1991); Green et al., Nature 320, 134 (1986); White et al., Mol. Endocr. 1, 735 (1987); and Piva et al., J. Steroid Biochem. Molec. Biol. 46, 531 (1993).
U.S. Pat. No. 6,162,606 is directed to identification of defective estrogen receptors associated with the classification of breast tumors which are responsive to or resistant to hormone therapy. Similarly, U.S. Pat. No. 5,563,035 regards monitoring the level of ERF-1, a transcriptional regulator of expression of the estrogen receptor, as being indicative of the response of a breast tumor to various therapies.
There is epidemiological evidence that there are genetic alterations that are closely associated with morphological tumor progression, such as is found in studies in colon carcinoma (Vogelstein and Kinzler, 1993). In this model (Dupont and Page, 1985), breast cancer is hypothesized as evolving from normal ductal epithelium to typical hyperplasia, to atypical hyperplasia, to carcinoma in situ, to invasive carcinoma, and finally to metastatic carcinoma. Recent data also suggests that the majority of hyperplasias share molecular alterations with invasive disease in the same breast (O""Connell et al., 1998), providing genetic evidence that they are related. Unlike colon cancer, very little is known about the specific molecular changes that are associated with the earliest stages of breast cancer evolution. However, it is likely that estrogens are important, since they are potent mitogens for normal breast epithelial cells, and it is believed that the duration of estrogen exposure to the breast epithelium is a significant risk factor for breast cancer development. It is also generally agreed that expression of the estrogen receptor (ER) is relatively low in normal breast epithelium, but is higher in certain premalignant lesions (e.g. typical hyperplasias) (van Agthoven et al., 1994).
Anandappa et al. (2000) detected no sequencing variants, such as single base change mutations, in ER from a panel of human primary breast cancer specimens. However, Zhang et al. (1997) identified an ER mutant in metastatic breast cancer which had a constitutive transactivation function independent of estradiol-binding.
Current human breast cancer management strategies utilize ER status as a predictive factor (McGuire, 1978; Burstein, 1982; Brooks et al., 1980; Degenshein et al., 1980; McGuire et al., 1975; McGuire, 1987; Elledge and McGuire, 1993; Gelbfish et al., 1988; Williams et al., 1987; Kohail et al., 1985; Donegan, 1992; Millis, 1980; McCarty et al., 1980), although none regard the specific mutation of the present invention. Present human breast tumor tissue specimens are subjected to both ligand-binding studies and immunohistochemical analyses to determine ER status (King et al., 1979; Shousha et al., 1989; Shousha et al., 1990). Thus, as has been acknowledged (see, for example, Roger et al., 2000), the art presently lacks a molecular marker for breast tissue, such as a premalignant lesion, which is at risk for breast cancer, particularly for invasive breast cancer, and also lacks a marker for the purpose of improving approaches to risk prediction and treatment strategies. Identification of a specific molecular marker for an altered ER as an early event in breast cancer evolution would be a significant advance in the field and would provide an ideal diagnosis tool for the detection of susceptibility to breast cancer and its subsequent prevention.
In an embodiment of the present invention there is an isolated estrogen receptor alpha nucleic acid sequence comprising an A908G mutation.
In another embodiment of the present invention there is an isolated estrogen receptor alpha amino acid sequence comprising a K303R substitution.
In an additional embodiment of the present invention there is a method of detecting susceptibility to development of breast cancer in an individual, comprising the steps of obtaining a sample from a breast of the individual, wherein the sample comprises a cell having an estrogen receptor alpha nucleic acid sequence; and assaying the nucleic acid sequence for an A908G mutation, wherein the presence of the mutation in the nucleic acid sequence indicates the individual has breast cancer. In a specific embodiment, the sample is from a premalignant lesion of the breast.
In an additional embodiment of the present invention there is a method of detecting susceptibility to development of invasive breast cancer in an individual, comprising the steps of obtaining a sample from a breast of the individual; and assaying an estrogen receptor alpha nucleic acid sequence from a cell of the sample for an A908G mutation, wherein the presence of the mutation in the nucleic acid sequence detects susceptibility of the premalignant lesion to develop into the invasive breast cancer. In a specific embodiment, the sample is from a premalignant lesion of the breast.
In an additional embodiment of the present invention there is a method of detecting susceptibility to development of invasive breast cancer from a premalignant lesion in a breast, comprising the steps of obtaining a sample from the premalignant lesion; dissecting the sample to differentiate hyperplastic cells in the sample from nonhyperplastic cells; and assaying an estrogen receptor alpha nucleic acid sequence from the hyperplastic cell of the sample for an A908G mutation, wherein the presence of the mutation in the nucleic acid sequence detects susceptibility of the premalignant lesion to develop into the invasive breast cancer. In a specific embodiment, the dissection step comprises removal of the hyperplastic cells from the sample by manual manipulation or by laser capture microdissection. In another specific embodiment, the sample is obtained by biopsy. In a specific embodiment, the assaying step comprises sequencing, single stranded conformation polymorphism, mismatch oligonucleotide mutation detection, or a combination thereof. In an additional specific embodiment, the assaying step is by antibody detection with antibodies to the A908G mutation of the estrogen receptor alpha nucleic acid sequence or is by antibody detection with antibodies to an acetylated estrogen receptor alpha amino acid sequence.
In an additional embodiment of the present invention there is a method of classifying breast cancer in an individual, comprising the steps of obtaining from the individual a sample from the breast, wherein the sample contains a cancer cell; and assaying an estrogen receptor alpha nucleic acid sequence from the cell of the sample for an A908G mutation, wherein the presence of the mutation identifies the breast cancer to be invasive breast cancer. In a specific embodiment, the sample is obtained by biopsy. In another specific embodiment, the assaying step is selected from the group consisting of sequencing, single stranded conformation polymorphism, mismatch oligonucleotide mutation detection, and a combination thereof. In an additional specific embodiment the assaying step is by antibody detection with antibodies to the A908G mutation of the estrogen receptor alpha nucleic acid sequence or by antibody detection with antibodies to an acetylated estrogen receptor alpha amino acid sequence.
In another embodiment of the present invention there is a method of diagnosing breast cancer in an individual, comprising the steps of obtaining a sample from a breast of the individual, wherein the sample comprises a cell having an estrogen receptor alpha nucleic acid sequence; and assaying the nucleic acid sequence for an A908G mutation, wherein the presence of the mutation in the nucleic acid sequence indicates the individual has breast cancer.
In another embodiment of the present invention there is a method of diagnosing breast cancer in an individual, comprising the steps of obtaining a sample from a breast of the individual; dissecting the sample to differentiate a cell suspected of being cancerous from a noncancerous cell; and assaying the cell suspected of being cancerous for an A908G mutation in an estrogen receptor alpha nucleic acid sequence, wherein the presence of the mutation in the nucleic acid sequence indicates the individual has breast cancer. In a specific embodiment, the dissection step comprises removal of the cells suspected of being cancerous from the sample by manual manipulation or by laser capture microdissection. In a specific embodiment, the sample is obtained by biopsy. In another specific embodiment, the assaying step is selected from the group consisting of sequencing, single stranded conformation polymorphism, mismatch oligonucleotide mutation detection, and a combination thereof. In an additional specific embodiment, the assaying step is by antibody detection with antibodies to the A908G mutation of the estrogen receptor alpha nucleic acid sequence or is by antibody detection with antibodies to an acetylated estrogen receptor alpha amino acid sequence.
In another embodiment of the present invention there is a kit for diagnosing an A908G mutation in an estrogen receptor alpha nucleic acid sequence, comprising at least one primer selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In one embodiment, the primers are extendable. In an alternative embodiment, the primers are nonextendable.
In another embodiment of the present invention there is a monoclonal antibody that binds immunologically to an acetylated estrogen receptor alpha amino acid sequence, or an antigenic fragment thereof.
In another embodiment of the present invention there is a monoclonal antibody that binds immunologically to an A908G mutation in an estrogen receptor alpha nucleic acid sequence.
In an additional embodiment of the present invention there is a method to correct a G mutation at nucleotide 908 of an estrogen receptor alpha nucleic acid sequence in a cell of an individual, comprising the step of administering to the cell an estrogen receptor alpha nucleic acid sequence comprising an A at nucleotide 908. In a specific embodiment, the estrogen receptor alpha nucleic acid sequence comprising an A at nucleotide 908 is present on a vector. In another specific embodiment, the vector is selected from the group consisting of plasmid, viral vector, liposome, and a combination thereof. In an additional specific embodiment, the viral vector is selected from the group consisting of adenoviral vector, retroviral vector, adeno-associated viral vector, or a combination thereof.
In an additional embodiment of the present invention there is a method to prevent breast cancer in an individual, comprising the steps of obtaining a sample from a breast of the individual; identifying in the sample an A908G mutation in a nucleic acid sequence of estrogen receptor alpha; and correcting the A908G mutation, wherein the correction results in the prevention of the breast cancer. In a specific embodiment, the breast sample is from a premalignant lesion of the breast. In another specific embodiment, the correction step comprises administering an estrogen receptor alpha nucleic acid sequence comprising a G at nucleotide 908 to a cell comprising an estrogen receptor alpha nucleic acid sequence containing the A908G mutation.
In an additional embodiment of the present invention there is a method to treat breast cancer in an individual, wherein an estrogen receptor alpha nucleic acid sequence in a breast cell of the individual has an A908G mutation, comprising the step of administering to the cell an estrogen receptor alpha nucleic acid sequence comprising a G at nucleotide 908.
In another embodiment of the present invention there is a method to prevent breast cancer in an individual, comprising the steps of obtaining a sample from a breast of the individual; identifying in the sample an arginine at amino acid residue 303 in an amino acid sequence of estrogen receptor alpha; and administering to the individual an amino acid sequence of estrogen receptor alpha comprising a lysine at amino acid residue 303, wherein the administration results in the prevention of the breast cancer. In a specific embodiment, the breast sample is from a premalignant lesion of the breast.
In an object of the present invention there is a method of identifying a modulator of an estrogen receptor alpha K303R polypeptide, comprising providing a candidate modulator; admixing the candidate modulator with an isolated compound or cell, or a suitable experimental animal; measuring one or more characteristics of the compound, cell or animal; and comparing the characteristic measured with the characteristic of the compound, cell or animal in the absence of the candidate modulator, wherein a difference between the measured characteristics indicates that the candidate modulator is the modulator of the compound, cell or animal.
In another object of the present invention, there is a method of screening for a modulator of an estrogen receptor alpha polypeptide comprising a K303R substitution, comprising introducing to a cell a vector comprising a nucleic acid sequence which encodes the estrogen receptor alpha K303R polypeptide; a vector comprising at least one estrogen-responsive regulatory element operatively linked to a reporter polynucleotide; and a test agent; and assaying expression of the reporter polynucleotide in the presence of the test agent, wherein the test agent is the modulator when the reporter polynucleotide expression changes in the presence of the test agent. In a specific embodiment, at least one of the vectors is transiently transfected into the cell. In another specific embodiment, at least one of the vectors is stably transfected into the cell. In an additional embodiment, when expression of the reporter polynucleotide is upregulated, the modulator is an agonist. In an additional embodiment, when expression of the reporter polynucleotide is downregulated, the modulator is an antagonist. In a further specific embodiment, when the expression of the reporter polynucleotide is downregulated, the modulator is an antagonist. In a specific embodiment, the cell is a mammalian cell. In a further specific embodiment, the mammalian cell is selected from the group consisting of CHO, HepG2, HeLa, COS-1, MCF-7, MDA-MB-231, T47D, ZR-75, MDA-MB-435, BT-20, MDA-MB-468, and HEC-1. In an additional specific embodiment, the estrogen-responsive regulatory element is selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49; SEQ ID NO:22; SEQ ID NO:26, and SEQ ID NO:8. In an additional specific embodiment, the reporter polynucleotide is luciferase, chloramphenicol acetyltransferase, renilla or xcex2-galactosidase. In a specific embodiment, there is a method of treating breast cancer in an individual comprising the step of administering the antagonist to the individual.
In another object of the present invention, there is a method of identifying a polypeptide which interacts with an estrogen receptor alpha polypeptide comprising a K303R substitution, comprising introducing to a cell, a vector comprising a polynucleotide which encodes a chimeric polypeptide comprising the estrogen receptor alpha K303R polypeptide and a DNA binding domain; introducing to the cell, a vector comprising a polynucleotide which encodes a chimeric polypeptide comprising a candidate polypeptide and a DNA activation domain; and assaying for an interaction between the DNA binding domain and the DNA activation domain, wherein when the interaction occurs, the candidate polypeptide is the polypeptide which interacts with the estrogen receptor alpha K303R polypeptide. In a specific embodiment, the polypeptide which interacts with the estrogen receptor alpha K303R polypeptide is an antagonist of the estrogen receptor alpha K303R polypeptide. In a specific embodiment, the interaction is assayed by assaying for a change in expression of a reporter sequence. In a specific embodiment, the cell is a yeast cell. In another specific embodiment, the cell is a mammalian cell. In a further specific embodiment, the DNA activation domain and the DNA binding domain are from GAL4 or LexA. In an additional specific embodiment, the reporter sequence is selected from the group consisting of xcex2-galactosidase, luciferase, chloramphenicol acetyltransferase, and renilla. In a specific embodiment, there is a method of treating an individual for breast cancer, comprising administering the antagonist to the individual.
In another object of the present invention, there is a method of identifying a peptide which interacts with an estrogen receptor alpha K303R polypeptide, comprising obtaining an estrogen receptor alpha K303R polypeptide having an affinity tag and a label; introducing the polypeptide to a substrate comprising a plurality of bacteriophage, wherein the bacteriophage produce a candidate peptide; and determining binding of the polypeptide with the candidate peptide, wherein when the polypeptide binds the candidate peptide, the candidate peptide is the interacting peptide. In a specific embodiment, the label is a color label, a fluorescence label, or a radioactive label. In another specific embodiment, the affinity tag is biotin, GST, histidine, myc, or calmodulin-binding protein.
In an additional object of the present invention, there is a method of identifying a compound for the treatment of breast cancer associated with an estrogen receptor alpha K303R polypeptide, comprising the steps of obtaining a compound suspected of having the activity; and determining whether the compound has the activity. In a specific embodiment, the compound having the activity is an antagonist of the estrogen receptor alpha K303R polypeptide. In a specific embodiment, the method further comprises dispersing the compound in a pharmaceutical carrier; and administering a therapeutically effective amount of the compound in the carrier to an individual having the breast cancer.
Another object of the present invention is the compound obtained by the method of identifying a compound for the treatment of breast cancer associated with an estrogen receptor alpha K303R polypeptide, comprising the steps of obtaining a compound suspected of having the activity; and determining whether the compound has the activity.
An additional object of the present invention is a pharmacologically acceptable composition comprising the compound obtained by the method of identifying a compound for the treatment of breast cancer associated with an estrogen receptor alpha K303R polypeptide, comprising the steps of obtaining a compound suspected of having the activity; and determining whether the compound has the activity; and a pharmaceutical carrier.
Other and further objects, features, and advantages would be apparent and eventually more readily understood by reading the following specification and be reference to the accompanying drawings forming a part thereof, or any examples of the presently preferred embodiments of the invention given for the purpose of the disclosure.